Hepatitis C Virus (HCV) efficiently establishes persistent infection, with 200 million people currently infected worldwide. In the US, chronic HCV infection accounts for 15,000 deaths via end-stage liver diseases such as liver cancer and decompensated cirrhosis. In addition, nearly 20,000 individuals are newly infected with HCV annually, thereby posing a continuous threat to public health. Thus, an effective prevention strategy remains essential to mitigating the burden of HCV. Towards this ultimate goal, furthering our understanding of host immunity against viral infection plays a critical role. Interferon stimulated genes (ISG) constitute over 300 innate immune effectors which cooperatively restrict viral infection and are critical determinants for efficient mounting of adaptive immunity. ISG expression is dynamic, occurring through differential strength and duration of interferon (IFN) signaling. However, the processes that regulate ISG expression to facilitate viral restriction are poorly defined. In search of novel host factors that stimulate ISG expression for the suppression of HCV infection, we have conducted comprehensive genome-wide cDNA screening. These studies identified the non-receptor tyrosine kinase 1 (TNK1) as a signaling molecule pivotal for enhanced ISG expression and restriction of HCV infection. Our studies indicate that TNK1 presides over a novel signaling pathway that imparts serine phosphorylation of STAT1, resulting in induction of a specific group of ISG that have potent anti-HCV activity. Therefore the proposed studies aim to investigate the hypothesis that the TNK1 pathway induces anti-HCV effectors through a unique process of STAT1 activation. We will conduct the following aims: (Aim 1) Determine the mechanism of TNK1 activation and characterize its enzymatic activity, (Aim 2) Define the signaling cascade governed by TNK1 mediated serine phosphorylation of STAT1, and (Aim 3) Determine the in vivo role of TNK1 in hepatic antiviral innate immunity. The results from this study will provide novel insights into a front line defense against viral infection and contribute to a prevention strategy against HCV infection and emerging viral diseases.